Inflammatory Processes
Inflammation is generally accompanied by changes in the metabolism of arachidonic acid, metabolism of nitric oxide, and creation of free radicals. Anti-inflammatory non-steroid drugs (NSAIDS), such as aspirin, can block certain links of an inflammatory process, but these drugs cannot stabilize damaged cellular membranes, which makes their influence on an inflammatory process limited and insufficient.
Inflammation is a localized reaction of live tissue due to an injury, which may be caused by various endogenous and exogenous factors. The exogenous factors include physical, chemical, and biological factors. The endogenous factors include inflammatory mediators, antigens, and antibodies. Endogenous factors often develop under the influence of an exogenous damage. An inflammatory reaction is inevitably followed by an altered structure and penetrability of the cellular membrane. At the tissue and organ level, inflammation is indicated by pain, swelling, reddening, increased temperature, and a lost function in some cases. Inflammation begins with a sub-lethal damage and terminates either with a complete recovery or long-term tissue ruination. There is no recovery from an injury without an inflammation.
An immediate response to a tissue damage is realized via mediators, which are released due to the exocytosis or lysis of cells. The main inflammatory mediators are compounds of the kinine and fibrinolytic systems, the complement system, metabolites of arachidonic acid, vasoactive amines, and other chemical compounds. The chemical mediators of inflammation include: histamine, serotonin, prostaglandins, CGRP, nitric oxide, among others.
An important role in inflammations is played by various reactive oxygen-containing species. These compounds are synthesized when oxygen transforms them into very dangerous forms, producing free radicals, which are atoms and molecules with unpaired electrons. Different free radicals have different activity levels.
The launch of an inflammation is influenced by various exogenous and endogenous agents. Endogenous factors, namely, mediators, antigens, and autogens define the nature and type of the inflammatory reaction, especially its course in the zone of injury. In the case where a tissue damage is limited to the creation of mediators, an acute form of inflammation develops. If immunologic reactions are also involved in the process, through the interaction of antigens, antibodies, and autoantigens, a long-term inflammatory process will develop. Various exogenous agents, for example, infection, injury, radiation, also provide the course of inflammatory process on a molecular level by damaging cellular membranes which initiate biochemical reactions.
Inflammatory processes rely on the metabolism of arachidonic acid, which converts to prostaglandines (PG), tromboxanes (TX), and leukotrienes (LT). Prostaglandines, tromboxanes, and leukotrienes are the main participants of all inflammatory processes. There are two known ways of arachidonic acid cascade. The first way leads to the creation of prostaglandines G2 and H2. This process is catalyzed by prostaglandin-cyclooxygenase. Cyclooxygenase catalyzes the production of PGA2, PGE2, PGD2, PGF2α, while tromboxane-synthesis with PGH2 produces tromboxane A2 (TXA2).
The cascade of metamorphoses of arachidonic acid, which is a product of membrane and phospholypase A2, is best known. Through its cyclogenase and lypoxygenase cascades, arachidonic acid turns into prostaglandins and leukotrienes, respectively. The cyclooxygenase way leads to the formation of two bio-active products: prostacycline (PGI2) and thromboxane (TXA2). These products are involved in many inflammatory effects: bronchoconstriction, vazodilation, vasoconstriction, platelet aggregation, analgesia, pyrexia, et al.
Another way of arachidonic acid metabolism with 5-lipoxygenase leads to the synthesis of leukotrienes: LTA4, LTB4, LTC4, LTD4, LTE4, and LTF4. These leukotrienes have a powerful anti-inflammatory and bronchoconstrictor action, and they play and important role in vascular penetrability. Besides, leukotrienes are known as potential chemotactic factors; they increase the migration of WBC and have a great influence on the slow-releasing substance of anafilaxis (SRS-A).
Prostaglandines can play an important role in the development of systemic inflammatory reactions. In rheumatic arthritis, large quantities of PG and LT in the synovial liquid support the development of an inflammatory process and demineralization of bone tissue surrounding joints. Leukotrienes are known to be the main patho-physiological mediators of inflammatory reactions. They influence, to a greater degree than prostaglandines, the penetrability of vessels and the adhesion of leukocytes to vessel walls as well as the development of edema.
Prostaglandines effectively regulate the aggregation of platelets. PGE1 is a powerful inhibitor of platelets aggregation, while PGE2, which is normally released from platelets, stimulates this process. However, the most important role in blood coagulability is played by PGI2, or prostacycline, which is synthesized in blood vessel walls by arachidonic acid. It is the most powerful inhibitor of platelets aggregation, which has vasodilator properties. Thromboxane, which is synthesized in platelets, has an opposite action.
When endothelium is damaged, the adhesion of platelets with subendothelium tissue and the aggregation of platelets is initiated. The main role in this process is played by thromboxane A2. Prostaglandin I2, on the contrary, inhibits the aggregation of platelets. Therefore, the proportion of PGI2 and TXA2 is crucial for the process of coagulation.
Further, a special role in the process of recovery from inflammation is played by nitrogen oxide (NO). This gas easily penetrates in different organs and tissues and, as a free radical, has a powerful reactivity. Nitrogen oxide is a potent vasodilator, neurotransmitter, and inflammatory mediator, which plays a significant role in asthmatic inflammation.
Nitrogen oxide is produced endogenously by L-arginine amino acid and NO-synthetase. There are three known forms of NO-synthetase, two of which are constituent, and one inducible. The inducible NO-synthetase, which is expressed in the epithelium cells, quickly increases its activity when anti-inflammatory cytokines (such as interleukin 1 beta (IL-1beta) and tumor necrosis factor (TNF-alfa) are released.
Nitrogen oxide has both positive and negative properties with respect to an inflammatory reaction. One important and potentially positive property is its ability to relax the smooth bronchial muscle, which results in bronchodilation. Its negative properties include the ability to help the inflammatory process by increasing chemotaxis neutrophils, monocytes, and oesinofils with the help of the guasine-monophosphate-dependent mechanism. It is believed that nitrogen oxide inhibits adhesion of leukocytes to vascular endothelium and bronchial epithelium.
NO plays an important biological role in defining basal vascular tonus, regulating contractions of myocardium, and modulating the interaction between thrombocytes and vascular walls (Zhou Q., Hellermann G. R., Solomonson L. P., Nitric oxide release from resting human platelets, Thromb. Res., 1; 77(1):87–86; 1995). The role of thrombocyte activation in the pathogenesis of various thrombo-vascular conditions in humans and evidence about decreased NO-mediated effects in hypertension (Calver A., Collier J., Moncada S., Vallance P., Effect of local intra-arterial NG-monomethyl-L-arginin in patients with hypertension: the nitric oxide dilator mechanism appears abnormal, J. Hypertens., 10(9):1025–1031; 1990), diabetes (Calver A., Collier J., Valance P., Inhibition and stimulation of nitric oxide synthesis in the human foream arterial bed of patients with insulin-dependent diabet, J. Clin. Invest., 90(6):2548–2554; 1992), and artherosclerosis (Drexler H., Zeiher A. M., Meinzer K., Just H., Correction of endotelial dysfunction in coronary microcirculation of hypercholesterolaemic patient by L-arginine, Lancet., 21–28; 338(8782–8783); 1546–1550; 1991) suggests that drugs which increase the activity of NO-synthetase may effectively be used in treatment of patients. Human thrombocytes are capable of synthesizing nitric oxide. Large quantities of nitric oxide, for example, in the cells of endothelium, may be produced by intact thrombocytes, as well as by stimulated thrombocytes. Hence, nitric oxide of thrombocyte origin plays an important role in the support of vascular homeostasis and other NO-sensitive processes. (Zhou et al., 1995).
Beside some common features, inflammatory processes in each individual case have certain distinctions related to the peculiarities of functioning of the body organ and to the factors which caused the impairment: i.e., viruses, microorganisms, injuries, poisoning, etc.
For example, one of the common mechanisms of heart diseases, including acute infarct myocarditis, is a malfunction of the structure and function of the membrane of heart cells As a result, the synthesis of leukotrienes, tromboxanes, etc., which have coronoconstrictor, arrythmogenic, hemoatractive and pro-aggregate action, increases (Bangham A. D., Hill M. W., Miller N., Preparation and use of liposom as model of biological membranes, Methods in Membrane Biology, Acad. Press, V. 1, N.Y., P. 1–68, 1974).
Another important factor in the pathogenesis of heart impairments is the coronoconstrictor and hemoattractive (with regard to neutrofiles) action of lipoxygenase derivatives LTC4, LTD4, LTB4 (Hoshida S, Kuzuya T., Nishida M., et al., Amer. J. Cardiol, 7; 63(10): 24E–2E; 1989; Lam B. K., Gagnon L., Austen K. F. et al., J. Biol. Chem., 15; 265(23): 13438–1341; 1990; Svendsen J. N., Hansen P. R., Ali S. et al., Cardiovasc. Res., 27(7): 1288–1294; 1993). Substances which can block this process can in turn reduce the size of necrosis at acute myocardial infarction and, therefore, significantly decrease the lethality in difficult cases of heart disease, such as gross myocardial infarction. At the same time, such substances can stabilize the membranes of heart cells. In addition, it is known that coronoconstrictor and hemoattractive effects during infarct are accompanied by an increased aggregation of platelets. Therefore, blocking this process also leads to a decrease of the size of impairment.
Further, disorders of the aggregate state of blood play an important role in the pathogenesis of various diseases. This is especially apparent in the pathogenesis of thrombo-vascular conditions in humans. It is known that a malfunction in the thrombo-vascular link of homeostasis is a key factor leading to disorders of the aggregate state of blood, by causing changes. in the Theological properties of, blood and triggering the formation of internal vascular aggregates. Thrombocyte-related injuries lead to failures in micro-circulation processes, which result in shortages of blood inflow to the tissue. At the initial stage of the formation of blood clots, platelets become activated and further undergo adhesion to the injured endothelium. Later on, they aggregate and an initial thrombocytic blood clot is formed.
Today, there is enough evidence of a close relation between inflammations, disorders in the aggregate state of blood, and cardiovascular conditions (Anderson J. L. Carlqist J. L., et al., Evaluation of C-reactive protein an inflammatory marker, and infectious serology as risk factors for coronary artery disease and myocardial infarction, J. Am. Coll. Card., 32: 35–41; 1998).
Damages to a cellular membrane or inflammatory processes in human body are often accompanied by blood cytopenia. In most cases, such patients have anemia, thrombocytopenia, or neutropenia. Anemia is accompanied by a decrease in the quantities of erythrocytes or hemoglobin, which is attributed to a blood loss, malfunction in the production of erythrocytes, increased destruction of erythrocytes, or to a combination of these causes. In the case of thrombocytopenia, the quantity of thrombocytes in blood is decreased, which causes a malfunction in thrombogenesis and subsequent bleeding. Neutropenia is a decrease in the count of neutrophiles in blood, which often leads to an increased sensitivity to various infections.
Normal formation of blood cells, or hematopoiesis, begins with a hematopoietic stem progenitor cell termed CFU-GEMM, which, in adults, is formed in the marrow and, under the influence of growth factors, is transformed in specialized blood cells. For example, erythrocytes are formed from CFU-GEMM under the influence of erythropoietin. If influenced by thrombopoietin, CFU-GEMM is transformed into thrombocytes. Similarly, under the influence of granulocyte-macrophage colony-stimulating factor, or CFU-GEMM is transformed into granulocytes and monocytes. Also, lymphocytes originate from a lymphoid stem cell.
The most pronounced cytopenia with severe consequences occurs in cancer patients, especially after chemotherapy and radiotherapy, in AIDS patients and those, infected with HIV (J. Crawford, J. L. Gabrilove, Therapeutic Option for Anemia and Fatigue, Medscape, Oncology Treatment Update, 2000, Medscape, Inc.).
Role of Cell Membranes in Inflammatory Processes
The functions of cell membranes and their relation to inflammatory processes has been documented. It is known that the plasmatic cellular membrane occupies a special place among the other membrane structures and performs such important functions as barrier and transportation, provides a contact with the outside environment for the cell, participates in the regulation of cellular homeostasis, supports signal mechanisms of this regulation, and defines the cell's individuality and wholeness. The structural organization, dynamics, and functions of erythrocytal membranes and various hemolysis patterns, such as osmotic, oxide, immune (induced by hemolytic viruses, toxins, complement), detergent hemolysis, photohemolysis, etc., are well studied (see, e.g., Bashford C. L., Alder G. M., Menestrina G., et al., Membrane damage by hemolytic viruses, toxins, complement, and other cytotoxic agents. A common mechanism blocked by divalent cation. J. Biol. Chem., 15; 261(20): 9300–9308, 1986; Osorie e Castro V. R., Ashwood E. R., Wood S. G., Vernon L. P., Hemolysis of erythrocytes and fluorescence polarization changes elicited by peptide toxins, aliphatic alcohols, related glycols and benzylidene derivatives, Biochim. Biophys. Acta., 16; 1029(2): 252–258; 1990).
It was demonstrated that pH variation in the outside environment upsets the balance of forces influencing the membrane, which leads to structural changes and changes of the aggregation degree of membrane proteins. Two types of membrane structural changes are distinguished: those caused by pH variation in the range 7.0–6.0, and those for pH levels below 4.5 (Zavodnik I. B., Pileckaya T. P., Acid lysis of human erythrocytes, Biophizika., V. 42, N. 5, P. 1106–1112, 1997). In the latter case, the membrane becomes destabilized and erythrocytal lysis follows. It is known that at pH 4.7, pores are formed in glycocalyx erythrocytal membranes (Arvinte T., Cudd A., Schulz B., Nicolau C., Biochim. Biophys. Acta., 19; 981(1): 61; 1989). In particular, decreased pH levels of the environment change the confirmation, package type, and mobility of phospholipids in model membranes. Thus, aggregation of membrane proteins, denatured due to a decreased pH, is the reason for membrane damages and acid lysis in erythrocytes.
The pattern of erythrocytal hemolysis by HCl was proposed based on the cooperative protonation of some center located in stroma or on the membrane of erythrocyte with a subsequent creation of pores, sufficient to release hemoglobin. By studying the mechanism and pattern of the acid hemolysis process, information about the structural organization of the membrane and membrane-stabilizing actions can be obtained.
The best known endogenous stabilizers of hemolysis in erythrocytes (osmotic hemolysis is the best-studied) are albumin of blood plasma, metallic ions K+, Na+, Mg2+ and, especially, Ca2+, which modulate the canals of plasmatic erythrocytal membranes, possibly including the proton canal (Anderson D. R., Davis J. L., Carraway K. L., Calcium-promoted changes of the human erythrocyte membrane. Involvement of spectrin, transglutaminase, and a membrane-bound protease. J. Biol. Chem., 10; 252(19): 6617–6623, 1977), cholesterol adsorbed on the surface of erythrocytes (Hui S. W., Stewart C. M., Carpenter M. P., Stewart T. P. Effects of cholesterol on lipid organization in human erythrocyte membrane, J. Cell. Biol., 85(2): 283–291; 1980), and polyamines, which bind with the fatty-acid residues of membrane phospholipids Rennert O. M., Shukla J. B., Polyamines in health and disease Advances in Polyamine research, Raven Press, V. 2, N.Y, P. 195–21, 1978). The best known activators of endogenous hemolysis in erythrocytes are long-chain fatty acids (Rybszynska M., Csordas A., Chain length-dependent interaction of free fatty acids with the erythrocyte membrane, Life Sci., 44(9): 625–632; 1989), and especially free radicals of oxygen and nitrogen (Sato Y., Kamato S., Takahashi T. et al., Mechanism of free radical-induced hemolysis of human erythrocytes: hemolysis by water-soluble radical initiator. 18; 34(28): 8940–8949; 1955; Sen T., Ghosh T. K., Chaudhuri A. K. Glucose oxidase-induced lysis of erythrocytes. J, Exp. Biol., 33; (1): 75–76; 1995; Wollny T., Yacoviello L. Propogation of bleeding time by acute hemolysis in rats: a role for nitric oxide. Am. J. Physiol. 272(6): 2875–2884; 1997).
In summary, there is evidence to suggest that the structure of the membrane is altered during inflammatory processes. However, the model of membrane damage in the inflammatory process has not been used for screening drugs and treating or preventing inflammation and inflammatory-related disorders.
Present Drugs Unsatisfactory
The present anti-inflammatory drugs are unsatisfactory because the difficult and various biochemical reactions involved in inflammations and the lack of reliable information about inflammatory pathogenesis complicate the experimental choice of pharmacological compounds capable to regulate inflammation. Thus, drugs are selected to have an effect on individual components of an inflammation. So far, there is no drug able to regulate most of the components of any inflammatory reaction.
Most of the known non-steroid anti-inflammatory drugs (NSAIDS) selectively influence certain phases of this pathological process. First, they influence the penetrability of blood vessels, which is often altered in acute inflammations, and various cell reactions, which are common for chronic inflammations. Also, many NSAIDS influence metabolism through the mechanism of free radicals.
The initial screening of anti-inflammation processes typically uses three groups of methods. First, the influence of drugs on easily-identifiable inflammatory symptoms is studied. These include swelling, hyperemia, necrosis, etc. A more advanced analysis includes experimental therapy methods, using model arthritis, carditis, etc., which are similar to human ailments. The third stage involves analysis of how the drug influences certain metabolic ways.
After the metabolism of arachidonic acid was studied in detail, many anti-inflammatory compounds, whose action was to regulate the formation of such metabolic products, were proposed. In most cases, such drugs act as inhibitors of the metabolic enzymes of arachidonic acid. One example is the anti-inflammatory pharmacological combination of cyclooxygenase 2 inhibitor and leukotriene A.sub.4 hydrolase inhibitor (Isakson, P. C., Anderson G. D., Gregory, S. A., Treatment of inflammation and inflammation-related disorders with a combination of a cyclooxygenase-2 inhibitor and a leukotriene A.sub.4 hydrolase inhibitor, U.S. Pat. No. 5,990,148, November 1999). A similar approach was proposed on the basis of analogues of pyrimidines, a component of nucleic acids (Connor D. T., Kostian C. R., Unangst P.C., 2-heterocyclic-5-hydroxy-1,3-pyrimidines useful as antiinflammatory agents, U.S. Pat. No. 5,240,929, August 1993). Since these compounds are the inhibitors of key metabolic ferments of arachidonic acid, 5-lipoxygenase and cyclooxygenase, the authors suggested their use as anti-inflammatory drugs suitable for treatment of a wide range of diseases, from allergenic conditions and rheumatoid arthritis to artheroscierosis and myocardial infarction. Other researchers recommended prostacyclin analogues for treatment of thrombocyte aggregation and bronchoconstriction (Haslanger M. F., Prostacyclin analogs and their use in inhibition of arachidonic acid-induced platelet aggregation and bronchoconstriction, U.S. Pat. No. 4,192,891, March 1980).
However, since an inflammatory process initiates many different metabolic cascades, the use of inhibitors or metabolic analogues of arachidonic acid does not allow to balance all such reactions and, hence, cannot regulate the complex inflammatory process in a satisfactory manner.
Further, aspirin, which has been used in applied medicine for a long time, has also been proposed since it can block metabolic ferments of arachidonic acid. Inhibitors of prostaglandines, such as aspirin, quite effectively influence the inflammatory processes. For this reason, they are successfully used in clinics for the treatment of rheumatoid arthritis, osteoarthritis, and other similar inflammatory processes. Aspirin also has anti-coagulation properties, since it inhibits the synthesis of TXA2, and it influences at least partially the synthesis of PGI2. A daily dose of 3 g of aspirin is commonly used for prevention of stenocardia, as a post-infarct and post-insult treatment, or for patients with a high risk of cardio-vascular conditions.
However, studies on the synthesis of TXA2 and PGI2 in vivo have shown that peroral administration of aspirin decreases the secretion of PGI2 only for 2–3 hours, while the secretion of thromboxane is halted for 10 days (Vesterqvist O., Measurements of the in vivo synthesis of thromboxane and prostacyclin in humans, Scand. J. Clin. Lab. Invest. 48(5): 401–407; 1988). This author, as well as others (see, e.g., Lorenz R. L., Boehlin B., Uedelhoven M. W., Weber P. C., Superior antiplatelet action of alternate day pulsed dosing versus split dose administration of aspirin, Am. J. Cardiol. 15; 64(18): 1185–1188; 1989), not only show the difficulties in administering the right dose of aspirin, but also provide and experimental ground for the frequent side effects caused by aspirin during its long-term use.
Specifically, aspirin and other non-steroid anti-inflammatory drugs may be the cause of anaphylactoid reactions in sensitive individuals. The mechanism of these reactions is dose-dependent toxic-idiosyncratic, not immunologic. Also, aspirin is the most common cause of accidental poisoning. Children, treated by aspirin before poisoning, are also at great risk. Aspirin overdose, which occurs frequently, is difficult to correct. The effective aspirin dose for many diseases, including rheumatoid arthritis, constitutes 3–6.5 mg per day, which leads to irritations of the gastro-intestinal tract. Patients with gastro-intestinal conditions do not tolerate aspirin. Aspirin also causes erosion, bleeding stomach ulcers, diarrhea, and duodenum ulcers. Further, aspirin is commonly used in treatment for its anti-thrombocytic action, but it is badly tolerated and causes side-effects when taken for a long period of time. In addition, by inhibiting non-selectively cyclooxygenesis, aspirin interferes with the synthesis of thromboxane, which is a powerful aggregant and vasoconstrictor, and may also lead to decreased levels of prostacycline, which is both anti-aggregant and vasodilator.
All these negative side-effects of aspirin and other NSAIDS motivate the search for new drugs which would have anti-inflammatory properties, but which are non-toxic in a wide range of concentration, have no side effects during a long-term use, and are capable of preventing and terminating inflammatory processes.
Further, a complex treatment of cytopenia is done with hematopoietic growth factors (J. Crawford, Hematopoietic Growth factor: Current Practice and Future Directions, 42-nd Annual Meeting of the American Society of Hematology, 2000, Medscape, Inc.). However, it is quite complicated and expensive. For example, the treatment of cytopenia in HIV-infected patients depends upon the specific cause of the abnormality, hematopoietic growth factors are used. Erythropoietin is used to treat anemia, thrombopoietin is used for thrombopenia, and G-CSF is used to treat neutropenia in HIV-infected patients. Thus, treatment of cytopenia in HIV-infected patients requires a very expensive diagnostics of the endogenous level of such growth factors and quite expensive growth factors, which are commonly obtained via recombinant technologies.
For this reason, a search of inexpensive drugs, which could normalize anemia, thrombocytopenia, and neutropenia is important.
Pharmaceutical Use of Nucleic Acids
Nucleic acids are commonly used in pharmacology (Rothenberg M., Jonson G., Laughlin C. et al. Oligodeoxynucleotides as anti-sense inhibitors of gene expression: therapeutic implications, J. Natl. Cancer Inst., 18; 81(20): 1539–1544; 1989; Zon G., Oligonucleotides analogues as potential chemotherapeutic agents, Pharm. Res., 5; (9): 539–549; 1988). However, pharmaceutical uses for nucleic acids have not included inflammatory or inflammatory-related disorders. For example, Anderson et al., proposes the method of modulating the effects of cytomegalovirus infections with the help of an oligonucleotide, which binds with mRNA of cytomegalovirus, for treatment of cytomegalovirus infections in humans (Anderson K., Draper K., Baker B., Oligonucleotides for modulating the effects of cytomegalovirus infections, U.S. Pat. No. 5,442,049, Aug. 15, 1995). On the basis of a specific nucleic acid, which encodes the succession of 3′ non-translated sector of protein kinase C, Boggs et al. propose a method for diagnosis and treatment of conditions, which are associated with protein kinase C alpha (Boggs R. T., Dean. N. M., Nucleic acid sequences encoding protein kinase C and antisense inhibition of expression thereof, U.S. Pat. No. 5,681,747, October 1997). Also, Yano et al. patented a DNA compound obtained from Mycobouterium bovis and Bacillus subtilis for treatment of stomach ulcers (Yano O., Kitano T., Method for the treatment of digestive ulcers, U.S. Pat. No. 4,657,896, April 1987).
In particular, it is known that ribonucleic acid (RNA), products of its partial hydrolysis, and synthetic poly-ribonucleotides have a wide range of bioactivity (Kordyum V. A., Kirilova V. S., Likhachova L. I., Biological action of exogenous nucleic acids, Visnyk ASC USSR, V. 41, N. 6, P. 67–78, 1977). They activate protein synthesis in cells (Sved S. C., The metabolism of exogenous ribonucleic acids injected into mice, Canad. J. Biochem., V. 43, N. 7, P. 949, 1965) and have anti-tumor activity (Niu M. C., Effect of ribonucleic acid on mouse acids cells, Sciens., N. 131, P. 1321, 1960). RNA can increase antibody generation and decrease the inductive phase of antibody genesis (Johnson A. G., Schmidtke I., Merrit K. et al., Enhancement of antibody formation by nucleic acids and their derivatives, in Nucleic acid in immunology, Berlin, P. 379, 1968; Merrit K., Johnson A. G., Studies on the adjuvant of bacterial endotoxins on antibody formation, 6. Enhancement of antibody formation by nucleic acids, J. Immunol., V. 94, N. 3, P416, 1965; Brown W., Nakono M., Influence of oligodeoxyribonucleotides on early events in antibody formation, Proc. Soc. Exper. Biol. Med., 5, V. 119, N. 3, P. 701, 1967). It was shown that certain increased or decreased immunologic indicators normalize under the influence of RNA. In the first place, this applies to T-lymphocytes, cooperation of T- and B-lymphocytes, activation of macrophage function, etc.
Further, exogenous RNA is used for the DNA synthesis in dividing cells and for the RNA synthesis in metabolizing cells. It was also determined that 2 hours after the introduction, exogenous RNA was included in the RNA of lymphocytes and macrophages (Enesco N. E., Fate of 14C-RNA infected into mice, Exper. Cell Res., V. 42, N. 3, P. 640, 1966). Evidence suggests that yeast tRNA can be included into cells in the form of intact molecules (Herrera F., Adamson R. H., Gallo R. C., Uptake of transfer ribonucleic acid by normal and leucemic cells, Proc. Nat. Acad. Sci. USA, 67(4): 1943–1950; 1970).
It was determined by analytical methods that RNA is present in practically all membranes of animal cells (membranes of endoplasmic reticulum, mitochondrial, nucleic, and plasmatic membranes). Its content, depending on the type of tissue and on the method of membrane isolation, varies between 0.5 and 4% of the dry weight of the membrane. Experimental results show that special membrane RNA exists in isolated membranes (Shapot V. S., Davidova S. Y., Liponucleoprotein as an integral part of animal cell membrans. Prog. Nucleic Acid Res. 11: 81–101; 1971; Rodionova N. P., Shapot V. S. Ribonucleic acid of the endoplasmatic reticulum of animal cells. Biochim et Biophis Acta, 24; 129(1); 206–209; 1966). The functions of membrane RNA are not fully understood.
The functions of membrane RNA in ribosome have been better studied. (Cundliffe E., Intracellular distribution of ribosoms and poliribosomes in Bacillus megaterriium. J. Mol. Biol., 28; 52(3): 467–481; 1970) Ribosomal RNA is contained in bacterial membranes, in the outer membranes of nuclei, inner and outer membranes of mitochondria, inner membrane of the Goldgi apparatus, which adjoins the plasmatic membrane, in the rugged endoplasmic reticulum, in different tissues in animals, humans, plants, microorganisms, and protozoa. It is possible that membrane glycolipids and glycoproteins, which contain N-acetylneuraminic acid, are involved in the formation of binding sites of ribosomal RNA in ribosomes, since membranes which are treated by neuronidase lose the ability to bind ribosomes. (Scott-Burden T., Hawtrey A. O., Preparation of ribosome free membranes from rat liver microsomes by means of lithium chloride. Biochem. J. 115(5): 1063–1069; 1969. Further, it is possible that binding sites of ribosomes and membranes are activated by the sexual hormones, and cancerogens damage this physiological mechanism. This conclusion is supported by decreased levels of membrane-bound RNA in the process of aging (Mainwaring W. J. The effect of age on protein synthesis in mouse liver. Biochem J. 113(5): 869–878; 1969) and after castration of animals (Tata J. R., The formation, distribution and function of ribosomes and microsomal membranes during induced amphibian metamorphosis. Biochem J. 105(2): 783–801, 1967). Extraction of spermine from a membrane leads to a separation of bound RNA from the membrane (Khawaja J. A. Interaction of ribosomes and ribosomal subparticles with endoplasmic reticulum membranes in vitro: effect of spermine and magnesium. Biochim. Biophis. Acta., 29; 254(1): 117–128); 1971). When membranes are treated with RNA of native small ribosomes of myeloma cells, they separate from the membranes, while large native subunits of ribosomes remain bound with the membranes (Mechler B., Vassalli P., Membrane-bound ribosomes of myeloma cells.I.Preparation of free and membrane-bound ribosomal fractions. Assessment of the methods and properties of ribosomes. J. Cell. Biol. 67(1): 1–15; 1975. Also, the nucleotide components of various membrane enzymes, for example, polyA-RNA enzyme of phosphofructokinase, constitute a possible pool of membrane RNA (Hofer H. W., Pette D. The complex nature of phosphofructokinase—a nucleic acid containing enzyme, Life Sci. 4(16): 1591–1596; 1965).
However, nucleic acids, and in particular RNA, and compositions containing the same, have not been used to treat or prevent inflammatory or inflammatory-related disorders. In particular, most of the studies above rely on experiments in vitro. Further, none of these methods is directed to treating or preventing an inflammation or inflammatory-related disorder.
Need for New Drug
In view of the above, there is a need for new anti-inflammatory drugs which would regulate disorders of the aggregate state of blood and would have less negative effects than aspirin and other NSAIDS. In particular, since an inflammatory process in the initial stage is followed by alterations in the structure and functions of the membrane in the many cells involved in the inflammatory process, drugs are needed which, not only regulate all the components of an inflammatory metabolic cascade, but also stabilize membrane structures and functions in the involved cells. In particular, since the traditional therapy has little effectiveness in extensive infarcts, which are complicated by the cardiogen shock, there is a need for new drugs capable of stopping the destruction of cardiomyocytes.